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High-throughput sequencing of a single chromosome: a moth W chromosome

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Abstract

Y and W chromosomes have mostly been excluded from whole genome sequencing projects. Due to the high amount of repetitive sequences they are ‘difficult’ to assemble and therefore need special treatment in the form of, e.g. adapted assembly programs, a range of different libraries, and accurate maps, if possible. A minimum requirement for these approaches is pure template DNA. We therefore microdissected the W chromatin of highly polyploid cells from the flour moth, Ephestia kuehniella, and used Roche/454 and Sanger sequencing to generate 72.6 Mbp of DNA sequence. Nominal coverage was 4.3× of the 16.7 Mbp of W chromosomal DNA. We used these data to assess the genetic content of the W chromosome. This approach allowed us to determine constituent families of transposable elements, microsatellites, and recent insertion sites of mitochondrial DNA. However, no conventional protein-coding gene has yet been found. The sequence collection is a rich source for the definition of W-specific PCR markers and the reconstruction of W chromosome loci, as a step towards full reconstruction of the chromosome.

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Abbreviations

BAC-FISH FISH:

Using bacterial artificial chromosomes as probes

CGH:

Comparative genomic hybridisation

FISH:

Fluorescence in situ hybridisation

MITEs:

Miniature inverted-repeat transposable elements

mtDNA:

Mitochondrial DNA

numt mtDNA:

Insert in nuclear DNA

SSR:

Simple sequence repeat

TE:

Tranposable element

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Acknowledgments

We thank J. Westermann and K. Kalies (Institut für Anatomie, Universität Lübeck, Germany) for access to and help with the Zeiss-PALM laser microdissection system. Dan Otto (Hinxton, UK), Alexie Papanicolaou (CSIRO, Canberra, Australia) and Krishnakumar Kandaswamy (Cologne, Germany) supported us with help and helpful suggestions on bioinformatics. Daisuke Kageyama (National Institute of Agrobiological Sciences, Tsukuba, Japan) shared his expertise on Wolbachia with us and supplied the primers for diagnostic PCR. The technical assistance of Conni Reuter (Lübeck) is gratefully acknowledged.

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Authors and Affiliations

  1. Zentrum für Medizinische Strukturbiologie, Institut für Biologie, Universität Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany

    Walther Traut & Enno Hartmann

  2. Department of Entomology, Max Planck Institute for Chemical Ecology, Hans-Knoell-Strasse 8, 07745, Jena, Germany

    Heiko Vogel & David G. Heckel

  3. Institute for Biochemistry I; Medical Faculty, University of Cologne, Joseph-Stelzmann-Straße 52, 50931, Köln, Germany

    Gernot Glöckner

  4. Leibniz Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 301, 12587, Berlin, Germany

    Gernot Glöckner

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  1. Walther Traut
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  2. Heiko Vogel
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  3. Gernot Glöckner
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  4. Enno Hartmann
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  5. David G. Heckel
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Correspondence to Walther Traut.

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Responsible Editor: Fengtang Yang

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Traut, W., Vogel, H., Glöckner, G. et al. High-throughput sequencing of a single chromosome: a moth W chromosome. Chromosome Res 21, 491–505 (2013). https://doi.org/10.1007/s10577-013-9376-6

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